Duplex DNA is copied continuously on one strand (leading), but discontinuously on the other (lagging) strand to form short segments called Okazaki fragments. Thus, lagging strand synthesis is a much more complex process. Failure to properly synthesize and process Okazaki fragments can lead to increased mutations, genomic instability, and replication failure. How lagging strand synthesis is coordinated in eukaryotes is poorly understood. Herpes simplex virus (HSV) is a genetically tractable and simple organism that encodes most, but not all, of the enzymes required for DNA synthesis. The overarching hypothesis guiding the studies proposed in this application is that synthesis and processing of Okazaki fragments require the coordination of HSV-encoded and cellular enzymatic activities. In the first aim, we will examine the requirements for coordination of the HSV-1 primase activity with its polymerase activity and for coordination of the helicase activity with primase activities of UL5/UL52/UL8 complex. A series of half-forked and forked DNA substrates that contain high efficiency sites for initiation of primer synthesis will examine the optimum orientation for loading UL5/UL52/UL8 for priming compared to that for translocation and fork unwinding, and for elongation of primers by polymerase. In the second aim, the conditions and factors that trigger the cycling of the processive HSV polymerase to new primers following termination of Okazaki fragments will be determined. In the third aim, the protein:protein and protein:DNA interactions that occur under the most efficient in vitro conditions for initiation, termination, or processing of Okazaki fragments will be determined. In addition, we will use a global proteomics approach to identify by mass spectrometry, cellular and viral proteins that specifically associate with replicating DNA. A novel approach to gently and specifically isolate plasmids that amplify in cells in an HSV origin-dependent manner is proposed. A better understanding of the steps required for HSV lagging strand synthesis could lead to novel strategies to disrupt the virus replication cycle. The simple model system also provides a unique opportunity to understand the formation and processing of lagging strand DNA in mammalian cells that is so vital to genomic stability.